The cell regulatory system constituted by the membrane proteins OX40 Receptor (indicated in the literature also as OX40R, OX-40, OX-40 Antigen, TNFRSF4, or CD134) and OX40 Ligand (indicated in the literature also as OX40L, glycoprotein gp34, ACT-4-L, TNFSF4, CD134 Ligand, or CD134L) has a prominent role in the regulation of immune responses as well as in the formation of secondary lymphoid tissue, similarly to other proteins belonging to the tumor necrosis factor ligand/receptor superfamilies (Gravestein L and Borst J, 1998; Weinberg A, 2002). Many evidences on these activities have been provided by clinical observations and by animal models, for example by gene targeting experiments (Chen A et al., 1999; Kopf M et al., 1999; Murata K et al., 2000).
OX40 Receptor (OX40R, from now on) is a cell surface antigen, member of TNFR family, transiently expressed following T cell receptor (TCR) engagement and acting as a co-stimulatory receptor. It is considered as a highly specific CD4+ or CD8+ activation marker for T cells, being often over-expressed in inflammation sites associated to immunological pathologies, such as in multiple sclerosis or rheumatoid arthritis, as well as in tumor-infiltrating lymphocytes and in the peripheral blood of animal models of graft-versus host disease.
OX40 Ligand (OX40L, from now on) is a transmembrane protein, originally identified as a protein stimulated by human T cell lymphotropic virus 1 infection and CD40 activation (Miura S et al., 1991), having structural similarity to TNF and capable of forming cell-bound or secreted trimers. It is present on activated, antigen-presenting B and T cells, as well as dendritic cells, vascular endothelium cells and other non-hematopoietic tissues (for example heart, skeletal muscle, and pancreas).
OX40L interacts with OX40R as a homotrimer with a high affinity (Kd=0.2-0.4 nM), and various binding assays have been tested on this system (Taylor L et al, 2002; Taylor L and Schwartz H, 2001; Al-Shamkhani A et al., 1997). However, no tridimensional structure has been solved so far, neither detailed structure-activity studies have been performed, in order to provide any further molecular details on the mechanism of OX40L-OX40R interaction.
The interaction between OX40L and OX40R has a co-stimulatory effect to OX40R-expressing effector T cells, leading to a more robust cell responses due to the up-regulation of the cytokine production by T helper cells (Th1 and Th2) and to an increased survival of memory T cells through the inhibition of activation-induced cell death. Confirming evidences were also obtained in the transgenic mice lacking a functional OX40L gene and in autoimmunity animal models, where it was demonstrated that blocking the OX40R-OX40L interaction or depleting OX40R-positive T cells reduces clinical signs of autoimmunity.
Moreover, OX40L induces, upon OX40R binding, the expression of several genes, including the C—C chemokine RANTES, confirming the results obtained in endothelium models where OX40R-OX40L system appears involved in the control of activated T cells extravasation (Kotani A et al., 2002).
Cumulatively, these expression and functional data raise the possibility that the signal transduction pathways regulated by OX40L-OX40R interactions may help to prolong antigen-specific proliferative responses or otherwise influence the persistence, differentiation or reactivation of effector/memory T cell populations.
The interest on OX40R-OX40L system is related to the fact that, even if the intracellular signaling mechanisms have not yet completely understood, the expression profile of OX40R makes this protein a peculiar target for CD4+ T cells mediated diseases in clinical settings, for example in multiple sclerosis, where it is necessary to delete auto-reactive T cells. The hypothesis is that the products modulating OX40R activity may not have the serious side effects of conventional immunosuppressive therapies for autoimmune diseases and transplant rejection, which target all T cells.
The therapeutic potential of modulating the interaction between OX40L and OX40R was recognized by in vivo generated results obtained with OX40L-targeted immunotoxins (Weinberg A et al., 1996), anti-OX40R antibodies (Bansal-Pakala P et al., 2001), anti-OX40L antibodies (Stuber E and Strober W, 1996; Yoshioka Y et al., 2000; Tsukada N et al., 2000), and OX40L-Ig fusion proteins (Higgins L M et al. 1999; Weinberg A et al., 1999). These compounds are intended either to antagonize OX40L-OX40R interaction (for preventing the accumulation of activated CD4+ T cells at inflammatory sites) or to activate OX40R (as in some other pathological conditions, such as cancer).
Various OX40R binding agent, being either agonist or antagonist of OX40R, have been disclosed in the prior art as having positive effects on immunization and cancer treatment (WO 95/21915; WO 95/21251; EP 978287; WO 99/42585; WO 02/66044; U.S. Pat. No. 6,312,700). However, only large molecule such as the OX40L whole extracellular domain or antibodies are actually disclosed as being effective OX40R binding agents. This is also due to the fact that no real structure-activity studies have been performed to characterize this interaction, neither reliable information can be inferred from the analysis of other TNF/TNFR protein structures (Bodmer J L et al., 2002).
Since known OX40R binding agents proved to be useful as therapeutic and diagnostic agents, it would be desirable to identify compounds which, maintaining the binding and OX40L-competing properties of the large molecules above mentioned, are easier to generate and formulate such as peptides or other small molecules.